Substituted β-amino acid derivatives useful as platelet aggregation inhibitors

ABSTRACT

Novel substituted amino acid derivatives are provided which inhibit platelet aggregation and which are useful in pharmaceutical compositions and methods of inhibiting platelet aggregation.

This application is a continuation-in-part of U.S. application Ser. No.08/221,913, filed Apr. 1, 1994, now abandoned, which is a divisional ofU.S. application Ser. No. 07/953,601 filed Oct. 6, 1992, issued as U.S.Pat. No. 5,344,957 which is a continuation-in-part of U.S. applicationSer. No. 07/866,933, filed Apr. 10, 1992, now issued as U.S. Pat. No.5,239,113 which is a continuation-in-part of U.S. application Ser. No.07/777,811 filed Oct. 15, 1991, now abandoned.

FIELD OF THE INVENTION

This invention pertains to substituted β amino acid derivatives whichinhibit platelet aggregation.

BACKGROUND OF THE INVENTION

Fibrinogen is a glycoprotein present as a normal component of bloodplasma, It participates in platelet aggregation and fibrin formation inthe blood clotting mechanism,

Platelets are cellular elements found in whole blood which alsoparticipate in blood coagulation. Fibrinogen binding to platelets isimportant to normal platelet function in the blood coagulationmechanism. When a blood vessel receives an injury, the platelets bindingto fibrinogen will initiate aggregation and form a thrombus, Interactionof fibrinogen with platelets occurs through a membrane glycoproteincomplex, known as gplIb/IIIa; this is an important feature of theplatelet function. Inhibitors of this interaction are useful inmodulating platelet thrombus formation.

It is also known that another large glycoprotein named fibronectin,possesses cell-attachment properties. Various relatively largepolypeptide fragments in the cell-binding domain of fibronectin havebeen found to have cell-attachment activity. (See U.S. Pat. Nos.4,517,686; 4,589,881; and 4,661,111). Certain relatively short peptidefragments from the same molecule were found to promote cell attachmentwhen immobilized on the substrate or to inhibit attachment when in asolubilized or suspended form. (See U.S. Pat. Nos. 4,578,079 and4,614,517).

In U.S. Pat. No. 4,683,291, inhibition of platelet function is disclosedwith synthetic peptides designed to be high affinity antagonists offibrinogen binding to platelets. U.S. Pat. No. 4,857,508 disclosestetrapeptides having utility as inhibitors of platelet aggregation.

Other synthetic peptides and their use as inhibitors of fibrinogenbinding to platelets are disclosed by Koczewiak et al., Biochem. 23,1767-1774 (1984); Plow et al., Proc. Natl. Acad. Sci. 82, 8057-8061(1985); Ruggeri et al., Ibid. 83, 5708-5712 (1986); Ginsberg et al., J.Biol. Chem. 260 (7), 3931-3936 (1985); Haverstick et al., Blood 66 (4),946-952 (1985); and Ruoslahti and Pierschbacher, Science 238, 491-497(1987). Still other such inhibitory peptides are disclosed in EP PatentApplications 275,748 and 298,820.

U.S. Pat. No. 4,879,313 discloses compounds useful as inhibitors ofplatelet aggregation having the formula: ##STR1## wherein x=6 to 10,

y=0 to 4,

z=H, COOH, CONH2 or Cl-6 alkyl, Ar=phenyl, biphenyl or naphthyl, eachsubstituted with 1 to 3 methoxy groups, or an unsubstituted phenyl,biphenyl, naphthyl, pyridyl or thienyl group, and Asp=aspartic acidresidue.

European Patent Application 372,486 discloses N-acyl β amino acidderivatives of the formula: ##STR2## and their salts. Said compounds areuseful for inhibiting platelet aggregation in the treatment ofthrombosis, stroke, myocardial infarction, inflammation andarteriosclerosis, and for inhibiting metastasis.

European Patent Application 381,033 discloses amidino or guanidinoarylsubstituted alkanoic acid derivatives useful for the treatment ofthrombosis, apoplexy, cardiac infarction, inflammation, arteriosclerosisand tumors.

European Patent Application 445,796 discloses acetic acid derivativeswhich have inhibitory action on the bonding of adhesive proteins toblood platelets as well as on blood platelet aggregation and cell-celladhesion.

European Patent Application 372,486 discloses N-acyl beta amino acidderivatives and their salts. Said compounds are useful for inhibitingplatelet aggregation in the treatment of thrombosis, stroke, myocardialinfarction, inflammation and arteriosclerosis and for inhibitingmetastasis.

SUMMARY OF THE INVENTION

In accordance with the present invention novel substituted β amino acidderivatives are provided which modulate and/or inhibit plateletaggregation. These novel inhibitor compounds can be represented by thefollowing chemical Formula I ##STR3## or pharmaceutically acceptablesalts thereof, wherein R₆ and R₇ are independently selected from thegroup consisting of hydrogen, lower alkyl radicals, lower alkenylradicals, aromatic hydrocarbon radicals, alicyclic hydrocarbon radicals,and arylalkyl radicals wherein the radicals are optionally substituted.

It is another object of the invention to provide pharmaceuticalcompositions comprising at least one compound of the Formula I whichcompositions are useful in inhibiting or modulating plateletaggregation.

It is still another object of the invention to provide a method forinhibiting or modulating platelet aggregation in a mammal in need ofsuch treatment comprising administering a compound of the Formula I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of the Formula I:##STR4## or pharmaceutically acceptable salts thereof, wherein R₆ and R₇are independently selected from the group consisting of hydrogen, loweralkyl radicals, lower alkenyl radicals, aromatic hydrocarbon radicals,alicyclic hydrocarbon radicals, and arylalkyl radicals, wherein theradicals are optionally substituted.

Exemplifying the invention are the following compounds:

2S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]butanoicacid;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(1,1-dimethylethyl)ester;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(phenylmethyl)ester;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-methyl ester;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-ethyl ester;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(3-propenyl) ester;

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-(1,1-dimethylethyl) ester; and

N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-phenylmethyl ester.

As utilized herein, the term "lower alkyl" alone or in combination,means an acyclic straight or branched hydrocarbon radical containingfrom 1 to about 10, preferably from 1 to about 8 carbon atoms and morepreferably 1 to about 6 carbon atoms. Examples of such radicals includemethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, pentyl, iso-amyl, hexyl, octyl and the like.

The term "lower alkenyl" refers to an unsaturated acyclic hydrocarbonradical containing at least one double bond. Such radicals contain fromabout 2 to about 10 carbon atoms, preferably from about 2 to about 8carbon atoms and more preferably 2 to about 6 carbon atoms. Examples ofsuitable alkenyl radicals include propylenyl, buten-1-yl, isobutenyl,pentenylen-1-yl, 2-2-methylbuten-1-yl, 3-methylbuten-1-yl, hexen-1-yl,hepten-1-yl, and octen-1-yl, and the like.

The term "alicyclic hydrocarbon radical" or "cycloalkyl" means a cyclichydrocarbon radical which is saturated or unsaturated containing 3 toabout 10 carbon atoms, and preferably from 3 to about 6 carbon atoms.Examples of suitable alicyclic hydrocarbon radicals include cyclopropyl,cyclopropylenyl, cyclobutyl, cyclopentyl, cyclohexyl,2-cyclohexen-1-ylenyl, cyclohexenyl and the like.

The term "aromatic hydrocarbon radical" or "aryl" as used herein meansan aromatic carbon ring system composed of one or more aromatic ringswhich system contains 4 to about 16 carbon atoms, preferably 6 to about12 carbon atoms, and more preferably 6 to about 10 carbon atoms.Examples of suitable aromatic hydrocarbon radicals include phenyl,naphthyl, biphenyl and the like.

The term "alkoxy", alone or in combination, means an alkyl ether radicalwherein the term alkyl is as defined above and most preferablycontaining 1 to about 4 carbon atoms. Examples of suitable alkyl etherradicals include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,iso-butoxy, sec-butoxy, tert-butoxy and the like.

The term "arylalkyl radical" as used herein refers to a lower alkylradical, as defined above substituted by an aryl as defined above.Benzyl and phenylethyl are examples of such "arylalkyl radicals".

The term "optionally substituted radicals" as used herein means each ofthe above described radicals optionally substituted by groups such asalkyl alkoxy, hydroxy, halo, amino, nitro, cyano, carbonyl and the like.

The term "pharmaceutically acceptable salt" refers to a salt prepared bycontacting a compound of formula I, with an acid whose anion isgenerally considered suitable for human consumption. Examples ofpharmacologically acceptable salts include the hydrochloride,hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate,lactate, maleate, oxalate, malate, succinate, and tartrate and citratesalts. All of these salts may be prepared by conventional means byreacting, for example, the appropriate acid with the correspondingcompound of Formula I.

Suitable pharmaceutically-acceptable base addition salts of compounds ofFormula I, include metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine.

The compounds of the present invention may be administered by anysuitable route, preferably in the form of a pharmaceutical compositionadapted to such a route and in a dose effective for the treatmentintended. Therapeutically effective doses of the compounds of thepresent invention required to treat or arrest progress of the medicalcondition are readily ascertained by one of ordinary skill in the art.

Total daily dose administered to a host in single or divided doses maybe in amounts, for example, from 0.001 to 100 mg/kg body weight dailyand more usually 0.01 to 10 mg/kg and most preferably 3 mg/kg. Dosageunit compositions may contain such amounts or submultiples thereof tomake up the daily dose.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diets, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy.

The compounds of the present invention may be administered orally,parenterally, by inhalation spray, rectally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare, conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols which are solid at ordinary temperaturebut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose lactose or starch. Such dosage forms may also comprise, innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more active pharmaceutical agents. When administered as acombination, the therapeutic agents can be formulated as separatecompositions which are given at the same time or different times, or thetherapeutic agents can be given as a single composition.

In the structures and formulas herein, the bond drawn across a bond of aring can be to any available atom on the ring.

The compounds in this invention can exist in various isomeric forms andall such isomeric forms are meant to be included. Tautomeric forms arealso included in the invention. Pharmaceutically acceptable salts ofsuch isomers and tautomers are meant to be included as well.

The compounds of the present invention may be prepared by standardsynthetic methods combined with methods analogous to solution phasepeptide synthesis [see: The Peptides: Analysis, Synthesis, Biology (E.Gross and J. Meienhofer, eds.), Vol. 1-5, Academic Press, New York)].

Five general synthetic sequences are outlined in Schemes I-V. ##STR5##Wherein W is selected from the group consisting of hydrogen, lower alkylradicals, lower alkenyl radicals, lower alkynyl radicals, alicyclichydrocarbon radicals and aromatic hydrocarbon radicals, wherein saidradicals are optionally substituted with hydroxyl, lower alkoxy, loweralkyl, halogen, nitro, amino, acyloxy, and phenyl and naphthyl which maybe optionally substituted with halogen, nitro, lower alkoxy, and loweralkyl; and

R₂ is selected from the group consisting of hydrogen, lower alkylradicals of 1 to about 6 carbon atoms, lower alkenyl radicals of 2 toabout 6 carbon atoms, and lower alkynyl radicals of 2 to about 8 carbonatoms, alicyclic hydrocarbon radicals of 3 to 6 carbon atoms, aromatichydrocarbon radicals; wherein said radicals are optionally substitutedwith hydroxyl, lower alkoxy, lower alkyl, halogen, nitro, cyano, azido,ureido, ureylene, amino, trialkylsilyl, alkylsulfonyl, phenylsulfonyl,trifluoromethyl, acetoxy, acetylamino, and benzoylamino; carbonyl,carboxyl derivatives, alkylsulfonyl amino, and phenylsulfonyl amino.

In Scheme I. The aminobenzamidine 1 (i.e., Z is hydrogen) is coupled toan alkanoic, alkenoic (both substituted or not) or alkynoic diacid. Anactivated form of the diacid is preferentially used. These activatedforms include anhydrides, internal anhydride, acid chloride or one ofthe various activated forms as described in Principles of PeptideSynthesis. Bodansky, 1984, Springer-Verlag, the disclosure of which ishereby incorporated by reference. A highly preferred procedure involvescondensation of an anhydride (e.g., succinic anhydride 2) with a salt ofaminobenzamidine 1. The reaction is best conducted in a polar solventsuch as methylene chloride, acetonitrile, dioxane, dimethylformamide,dimethylsulfoxide or a mixture of such solvents in the presence of anacid binding agent such as sodium, potassium or cesium carbonate,triethylamine, pyridine, sodium hydride, dimethylaminopyridine,diazabicycloundecene, or a mixture of such agents, at temperaturesranging between 0° C. and 120° C. The final compounds are obtained bycoupling of the amidine derivative 3 with a properly protectedaminoacid. The amide bonds are formed using standard coupling reagents,e.g., dicyclohexylcarbodiimide (DCC), carbonyldiimidazole (CDI),disuccinimidyl carbonate (DSC),benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) or isobutyl chloroformate (mixed anhydride method). When the aminoacid used in the coupling was protected as an ester of the carboxylicacid function (4, W=alkyl, aryl, . . . ), the free acids 5 are obtainedby a suitable deprotection method as described by T. H. Greene in"Protective Group in Organic Synthesis", Wiley-Interscience, 1980, thedisclosure of which is hereby incorporated by reference. ##STR6##

W and R² have the values described in Scheme I and

A is selected from the group consisting of lower alkyl radicals, loweralkenyl radicals, lower alkynyl radicals and alicyclic radicals, whereinsaid radicals are optionally substituted with hydroxy, lower alkoxy,lower alkyl, halogen, alkoxycarbonylalkyl, amino, alkylamino,dialkylamino, acylamino, alkylthio, sulfonyl and aromatic hydrocarbonswhich are optionally substituted with halogen, nitro, lower alkoxy andlower alkyl.

Alternatively, an aminobenzonitrile 6, can be used for condensation withthe desired diacid or diacid derivative. In that case, the nitrile canbe converted to the amidine directly or at a later stage. When theaminobenzonitrile is used in the condensation reaction (Scheme II), thecyano group of the resulting intermediate 7 is converted to the amidine8 via the thioimidate in nearly quantitative yield. The thioimidate isformed by first treating the cyano compound with hydrogen sulfide (H₂ S)followed by alkylation with methyl iodide. Next, treatment of thethioimidate with ammonium acetate affords the amidine as the salt (HI).Alternatively, the nitrile 7 can be converted to the amidine 8 by theuse of lithium bis(trimethylsilyl)amide in an inert solvent such asdiethyl ether [R. T. Boere et al, J. Organomet. Chem., 331, 161-67,(1987)], the disclosure of which is hereby incorporated by reference.The desired compounds are obtained by coupling of the amidine derivative8 with a properly functionalized aminoacid. The amide bonds are formedusing standard coupling reagents as described above for Scheme I.##STR7##

Scheme III illustrates the preparation of derivatives using the aminonitriles as reagents. The cyano group is kept unchanged as a precursorfor the amidine function throughout two amide bond forming steps. Thefirst intermediate 10 is directly engaged in a reaction with the desiredamino acid. The intermediate 10 is then converted to the benzamidine. Amethod of choice to produce the amidine function is via the thioimidateprocedure as described in Scheme II. It is desirable, in Scheme III, toprepare the intermediate 11 as an ester. The most desirable ester is thet-butyl ester which can be deprotected to the acid by contact with astrong acidic medium as HBr/AcOH or trifluoroaceticacid/dichloromethane. ##STR8##

SCHEME IV and V. Substituted aminonitrile can be used to preparesubstituted N-aminobenzamidine succinyl derivatives as specificallyillustrated in Scheme IV for the chloro derivative 14. The beta aminoacids can be either purchased or prepared from commercially availablestarting materials using known methods as illustrated in Scheme V. Theracemic beta aryl beta amino acids can be prepared from the appropriatearylaldehyde, malonic acid, and ammonium acetate as shown in SchemeV--method 1 (Johnson and Livak J. Am. Chem. Soc. 299 (1936)]. Theracemic beta alkyl beta amino acids can be prepared from thecorresponding alkene and chlorosulfonyl isocyanate (CSI) which goesthrough the beta lactam intermediate as shown in Scheme V--method 2 [W.A. Szabo Aldrichimica Acta (1977); R. Graf Angew. Chem. Internat. Edit.172 (1968)]. The beta lactam can be opened to the ethyl ester bytreatment with anhydrous hydrochloric acid in ethanol as shown in SchemeV. For example, 1,3-butadiene and 3-phenyl-1-propene reacted with CSIforms the beta lactam which is subsequently followed by opening withanhydrous HCl in ethanol. An alternative method to form racemic betaamino esters is shown in Scheme V method 3. Nucleophiles can be added to4-benzoyloxy-2-azetidinone to afford a variety of 3-substituted betaamino esters after treatment with anhydrous HCl in ethanol. For example,1-lithio-2-trimethylsilylethyne is added to 4-benzoyloxy-2-azetidinoneto afford a beta amino ester after ring opening [for a similar reactionsee: D. H. Hua and A. Verma Tetrahedron Lett. 547-550 (1985) or T.Kametani, Heterocycles Vol. 17 463 (1982)]. As another example,4-benzoyloxy-2-azetidinone was reacted with allyltrimethylsilane underLewis acid catalysis [titanium tetrachloride-K. Prasad et al., Vol. 19Heterocycles 2099 (1982)]. The cyclopropyl derivatives are prepared fromthe corresponding vinyl compounds by treatment with diazomethane andpalladium acetate [U. Mande et al., Tetrahedron Lett. 629 (1975)] asshown in scheme V method 4. The racemic beta amino acids can be resolvedusing classical methods as described in the literature [E. Fischer, H.Scheibler, R. Groh Ber. 2020 (1910); E. Fischer, H. Scheibler Annalen337 (1911)].

Chiral beta amino acids can be prepared using many different approachesincluding the following methods: homologation of the alpha amino acidsusing an Arndt-Eistert reaction as shown in Scheme V method 5 [Meier andZeller Angew. Chem. Int. Ed. Eng. 32-43 (1975)] as shown in Scheme Fmethod 3 [M. Rodriguez et al Tetrahedron Lett. 5153 (1990); W. J.Greenlee J. Med. Chem. 434 (1985) and references therein]; fromenantiomerically pure precursors obtained from L-aspartic acid [i.e.,Scheme V method 6, see: M. Rodriguez Tetrahedron Lett. 923 (1991)];through the addition of chiral amines to alpha, beta unsaturated estersbearing a chiral auxiliary as shown in Scheme V method 7 [J. d'Angeloand J. Maddaluno J. Am. Chem. Soc. 8112-14 (1986)]; through anenantioselective hydrogenation of a dehydroamino acid as shown in SchemeV method 8 [see: Asymmetric Synthesis, Vol. 5, (J. D. Morrison, ed.)Academic Press, New York, 1985]; through the addition ofenantiomerically pure amines to alpha, beta unsaturated esters as shownin Scheme V method 9 [see: S. G. Davies and O. IchiharaTetrahedron:Asymmetry 183-186 (1991)].

Method 6 of Scheme V was used to obtain a versatile enantiomericallypure aldehyde intermediate. The aldehyde was reacted with methoxylamineto form an oxime. The appropriate organometallic was added to thealdehyde to afford the corresponding alcohol.

Purification of final compounds is by reverse phase high performanceliquid chromatography (High Performance Liquid Chromatograhy Protein andPeptide Chemistry, F. Lottspeich, A. Henscher, K. P. Hupe, eds. WalterDeGruyter, New York, 1981, the disclosure of which is herebyincorporated by reference) or crystallization.

Contemplated equivalents of the general formulas set forth above for theplatelet aggregation inhibitors and derivatives as well as theintermediates are compounds otherwise corresponding thereto and havingthe same general properties wherein one or more of the various R groupsare simple variations of the substituents as defined therein, e.g.,wherein R is a higher alkyl group than that indicated. In addition,where a substituent is designated as, or can be, a hydrogen, the exactchemical nature of a substituent which is other than hydrogen at thatposition, e.g., a hydrocarbyl radical or a halogen, hydroxy, amino andthe like functional group, is not critical so long as it does notadversely affect the overall activity and/or synthesis procedure.

Either the reactions can be successfully performed by conventionalmodifications known to those skilled in the art, e.g., by appropriateprotection of interfering groups, by changing to alternativeconventional reagents, by routine modification of reaction conditions,and the like, or other reactions disclosed herein or otherviseconventional, will be applicable to the preparation of the correspondingcompounds of this invention. In all preparative methods, all startingmaterials are known or readily preparable from known starting materials.

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. Those skilled in theart will readily understand that known variations of the conditions andprocesses of the following preparative procedures can be used to preparethese compounds. All temperatures expressed are in degrees centigrade.

Within the foregoing synthetic description and examples which follow,abbreviations have the following meanings:

CHCl₃ =chloroform

DMF=dimethylformamide

DMSO=dimethylsulfoxide

g=gram

MeOH=methanol

min=minute

h=hour

mol=mole

mmol=mmole

MW=molecular weight

TLC=thin layer chromatography

NMM=N-methylmorpholine

RPHPLC=Reverse Phase High Performance Liquid Chromatography

TDA-1=Tris[2-(2-methoxyethoxy)ethyl]amine

PTC=Phase Transfer Catalysis

mL=milliliter

EXAMPLE 1 Preparation of4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutoanoic acid ##STR9##

Aminobenzamidine di-HCl (25 g, 120 mmol), which is commerciallyavailable from Aldrich, was added to dry DMF (100 mL). To this was addeddry pyridine (100 mL) and succinic anhydride (12 g, 120 mmol) followedby N,N-dimethylaminopyridine (DMAP, 0.15 g). The product precipitatedafter heating for 0.5 hour at 100° C. The product was filtered, washedwith water, acetonitrile and ether. The white solid was suspended indioxane, 4N HCl in dioxane (100 mL) was added and the suspension stirredfor 1 hour, filtered and dried in a desiccator to give 28 g, 88%isolated yield of a yellowish-white solid whose nmr and mass spectra isconsistent with 4-[[4-(aminoiminomethyl)phenyl)amino]-4-oxobutanoic acidHCl salt. Mp: 270°-290° C., decomp.

EXAMPLE 23S-[[[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-(methoxymethylamino)-4-oxobutanoicacid ##STR10## Step 1. Preparation of3S-amino-4-(methoxymethylamino)-4-oxobutanoic acid

To N-tBoc-L-aspartic acid, beta-benzyl ester (10 g, 31 mmole) dissolvedin 50 mL methylene chloride was added triethylamine (4.31 mL, 31 mmole).To this was added benzotriazol-1-yloxytris(dimethylamino)-phosphoniumhexafluorophosphate (BOP) (10.8 g, 31 mmole). After several minutesO,N-dimethylhydroxyl amine hydrochloride (3.40 g, 33.5 mmole) andtriethylamine (4.31 mL, 32.3 mmole) were added and the reaction allowedto proceed for several hours. The reaction mixture was diluted to 200 mLby addition of methylene chloride and washed successively with diluteaqueous hydrochloric acid, saturated aqueous sodium hydrogen carbonate,and saturated aqueous sodium chloride solution. The organic layer wasdried with magnesium sulfate and volatiles removed in vacuo to give thecrude product. This product was dissolved in ethyl acetate and passedover a 4×4 cm pad of Merck 60 silica gel. The ethyl acetate wasevaporated to give 8.9 g of a product whose nmr and mass spec whereconsistent for desired product (78%).

The N-BOC amido benzyl ester prepared above (7.9 g, 21.6 mmole) wasdissolved in 50 mL methanol. The solution was transferred along with 0.5gm 3% palladium on carbon catalyst to a medium pressure hydrogenationapparatus equipped with a magnetic stirring bar, pressure gauge and gasinlet and outlet valves. Hydrogen was introduced (54 psig) and thereaction allowed to continue overnight. The catalyst was removed byfiltration over a celite pad and the solvent removed in vacuo to givethe desired N-BOC amido acid: ¹ H NMR (300 MHz, d₆ DMSO); 1.45 (s, 9H),2.8 (m, 2H), 3.20 (s, 3H), 3.72 (s, 3H), 4.55 (m, 1H). FABMS 283 (M+Li).

The N-BOC amido acid prepared above was dissolved in a minimum of1,4-dioxane and 50 mL of 4N HCl in dioxane added at room temperature.The reaction was allowed to proceed until no further gas evolution wasnoted (30 minutes) and the solvent evaporated. The desired amino acid asthe trifluoroacetate salt was isolated by preparative C-18 reverse-phasehigh-performance liquid chromatography (RPHPLC) and lyophilized to givea white powder (2.33 grams, 8 mmole, 38% overall isolated yield):

¹ H NMR (300 MHz, d₆ DMSO):3.02 (bs, 2H), 3.125 (s, 3H), 3.687 (s, 3H),4.183 (m, 1H). FABMS 177.1 (M+H).

Step 2. Preparation of3S-[[[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-(methoxymethylamino)-4-oxobutanoic acid.

Coupling of 3S-amino-4-(methoxymethylamino)-4-oxobutanoic acid to4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid was achieved inthe following fashion.4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid hydrochloride(0.70 g, 2.6 mmole) prepared in Example 1 was reacted withisobutylchloroformate (0.34 mL, 2.6 mmole) and an equivalent ofN-methylmorpholine (0.29 mL, 2.6 mmole) in DMF. Following activation3S-amino-4-(methoxymethylamino)-4-oxobutanoic acid (0.5 g, 1.7 mmole)was added with an equivalent of N-methylmorpholine and the reactionallowed to proceed overnight. The solvent was removed and the productisolated by preparative hplc and the fractions containing desiredproduct taken to pH 6 with lithium hydroxide. The lithium salt wasisolated by lyophilization. ¹ H NMR (300 MHz, d₆ DMSO): 2.65 (bm, 6H)3.05, 3.10 (s, 3H), 3.65, 3.7 (s, 3H), 7.8 (m, 5H), 9.5 (bd, 4H), 10.6(d, 1H). FABMS 394 (M+H), 400.3 (M+Li); HRFABMS.

EXAMPLE 3N-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid##STR11##

3S-[[[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-(methoxymethylamino)-4-oxobutanoicacid (1.6 g) from Example 2 was taken up in 300 mL water made acidic (pH2) with trifluoroacetic acid and heated at 60° C. for several hours. Theproduct diacid-benzamidine was isolated by preparative RPHPLC andlyophilized to give the desired product (0.84 g). ¹ H NMR (300 MHz, d₆DMSO): 2.55 (bm, 6H), 4.5 (m, 1H), 7.8 (s, 4H), 8.2 (d, 1H), 9.15 (bs,4H), 10.4 (d, 1H), FABMS 351.3 (M+H).

EXAMPLE 4 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid4-(1,1-dimethylethyl)ester ##STR12##

To a flame-dried flask under argon atmosphere,4-[[4-(aminoiminomethyl)phenyl]amino]-5-oxopentanoic acid (2.0 g, 7.4mmole) prepared in Example 1 is added to dry DMF and dry pyridine (1:1v/v, 70 mL) followed by N-methylmorpholine (0.74 g, 7.4 mmole) andN,N'-disuccinimidyl carbonate, DSC (1.9 g, 7.4 mmole) along with DMAP(50 mg) at room temperature. Upon cessation of gas evolution a solutionof L-aspartic acid beta-t-butyl ester (1.5 g, 8 mmol, obtained fromBACHEM) in DMF:pyridine (1:1, 20 mLs) is added and the reaction allowedto proceed overnight. The solvent is removed at 55° C. under reducedpressure and the product is purified by preparative C-18 reverse-phasehigh-performance liquid chromatography, RPHPLC (gradient of water and0.05% v/v trifluoroacetic acid (TFA) to water: TFA/acetonitrile and0.05% v/v TFA) and lyophilized to give the desired product as a TFAsalt.

EXAMPLE 5 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(phenylmethyl)ester ##STR13##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(phenylmethyl)ester is achieved using the procedure of Example 4 butsubstituting L-aspartic acid beta-benzyl ester (1.8 g, 8 mmol, Fluka)for L-aspartic acid beta-t-butyl ester. The coupling reaction is allowedto proceed overnight, volatiles are removed under reduced pressure andthe desired product is purified by rphplc and lyophilized to obtainsubstantially pureN-[4-[[4-(aminoiminomethyl)-phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(phenylmethyl)ester.

EXAMPLE 6 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-methyl ester ##STR14##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-methyl ester is achieved using the procedure of Example 4 butsubstituting L-aspartic acid beta-methyl ester (1.2 g, 8 mmol, Sigma)for L-aspartic acid beta-t-butyl ester. The coupling reaction is allowedto proceed overnight, volatiles are removed under reduced pressure andthe desired product is purified by RPHPLC and lyophilized to obtainsubstantially pureN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-methyl ester.

EXAMPLE 7 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-methyl ester ##STR15##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-ethyl ester is achieved using the procedure of Example 4 butsubstituting L-aspartic acid beta-ethyl ester hydrochloride (1.6 g, 8mmol, Aldrich) for L-aspartic acid beta-t-butyl ester. The couplingreaction is allowed to proceed overnight, volatiles are removed underreduced pressure and the desired product is purified by RPHPLC andlyophilized to obtain substantially pureN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-ethyl ester.

EXAMPLE 8 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(3-propenyl) ester ##STR16##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(3-propenyl) ester is achieved using the procedure of Example 4 butsubstituting L-aspartic acid beta-allyl ester (1.4 g, 8 mmol) forL-aspartic acid beta-t-butyl ester. The coupling reaction is allowed toproceed overnight, volatiles are removed under reduced pressure and thedesired product is purified by RPHPLC and lyophilized to obtainsubstantiallly pureN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,4-(3-propenyl) ester.

EXAMPLE 9 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-(1,1-dimethylethyl) ester ##STR17##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-(1,1-dimethylethyl) ester is achieved using the procedure of Example 4but substituting L-aspartic acid alpha-t-butyl ester hydrochloride (1.8g, 8 mmol, BACHEM or Nova Biochem) for L-aspartic acid beta-t-butylester. The coupling reaction is allowed to proceed overnight, volatilesare removed under reduced pressure and the desired product is purifiedby RPHPLC and lyophilized to obtain substantially pure N-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-(1,1-dimethylethyl) ester.

EXAMPLE 10 Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-phenylmethyl ester ##STR18##

Preparation ofN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-phenylmethyl ester. is achieved using the procedure of Example 4 butsubstituting L-aspartic acid alpha-benzyl ester hydrochloride (2.1 g, 8mmol, BACHEM or Nova Biochem) for L-aspartic acid beta-t-butyl ester.The coupling reaction is allowed to proceed overnight, volatiles areremoved under reduced pressure and the desired product is purified byRPHPLC and lyophilized to obtain substantially pureN-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]aspartic acid,1-phenylmethyl ester.

EXAMPLE 11 Dimethyl3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]pentanedioate##STR19## Step 1. Preparation of dimethyl-3-aminoglutarate

Dimethyl-3-oxoglutarate (10 g, 57 mmol) was added to methanol (225 ml)followed by ammonium formate (36 g, 570 mmol) and NaBH₃ CN (3.7 g, 57mmol) at 25° C. After 24 hours the methanol was removed in vacuo toleave a white mass. Methylene chloride was added and the mixturefiltered. The methylene chloride was evaporated resulting in an oilwhich was dissolved in 1N HCl (200 ml) and extracted with ether (100ml). The ether layer was discarded and the aqueous layer was made basicusing solid K₂ CO₃. The product was extracted into methylene chloridedried over Na₂ SO₄ and evaporated to give dimethyl-3-aminoglutarate (7.5g). ¹ H NMR (d₆ -DMSO) δ1.76 (bs, 2H), 2.45 (dd, 4H, J=8.08 Hz, 16.64Hz), 3.69 (s, 6H), 5.45 (m, 1H); MS (FAB) m/e 176.0 (M+H+).

Step 2. Preparation of dimethyl3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]pentanedioate

4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid hydrochlorideprepared in Example 1 (4.6 g, 17 mmol) was added to dry DMF (225 ml)followed by N-methylmorpholine (1.2 g, 17 mmol) and isobutylchloroformate (2.3 g, 17 mmol) at 25° C. The mixture was stirred for 5minutes. Dimethyl-3-aminoglutarate (3.0 g, 17 mmol) was added followedby dimethylaminopyridine. After 1 hour the solvent was removed underreduced pressure and the product purified by reverse phasechromatography (water/acetonitrile) to result in 3.5 g of a white solid:¹ H NMR (d₆ -DMSO) δ 2.37 (t, 2H, J=7.3 Hz), 2.55 (m, 2H), 2.57 (t, 2H,J=7.1 Hz), 3.57 (s, 6H), 4.35 (m, 1H), 7.79 (s, 4H), 7.99 (d, Hz), 9.1(bs, 2H), 9.19 (bs, 2H), 10.42 (s, 1H); MS (FAB) m/e 393.2 (M+H+).

Elemental Analysis Required for C₁₈ H₂₄ N₄ O₆.F₃.C₂ O₂ H.H₂ O: C, 47.42;H, 4.91; N, 11.14. Found: C, 47.12; H, 4.97; N, 10.99.

EXAMPLE 123-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]pentanedioicacid, monomethylester ##STR20##

Dimethyl3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]pentanedioateprepared in Example 11 (700 mg) was added to water/acetonitrile (20 ml)followed by lithium hydroxide (100 mg) at 25° C. The mixture was stirredfor 30 minutes. The course of the reaction was monitored by RPHPLC.After satisfactory monoester was formed the reaction was neutralizedwith TFA and purified by reverse phase chromatography(water/acetonitrile) to result in 460 mg white solid: ¹ H NMR (d₆ -DMSO)δ2.39 (t, 2H, J=7.3 Hz), 2.55 (m, 2H), 2.57 (t, 2H, J=7.1 Hz), 3.57 (s,3H), 4.32 (m, 1H), 7.78 (s, 4H), 7.99 (d, 1H, J=8.1 Hz), 8.92 (bs, 2H),9.16 (bs, 2H), 10.39 (s, 1H); MS (FAB) m/e 379.2 (M+H+).

Elemental Analysis Required for C₁₇ H₂₂ N₄ O₆.F3.C₂ O₂ H.H₂ O: C, 45.92;H, 4.63; N, 11.28. Found: C, 45.88; H, 4.34; N, 10.69.

EXAMPLE 13(±)-Diethyl-3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-4-dioxobutyl]amino]heptanedioate##STR21## Step 1. Preparation of diethyl-3-aminopimaleate

Diethyl-3-oxopimaleate (10 g, 43 mmol) was added to methanol (225 ml)followed by ammonium formate (27.4 g, mmol) and NaBH₃ CN (2.7 g, 43mmol) at 25° C. After 24 hours the methanol was removed in vacuo toleave a white mass. Methylene chloride was added and the mixturefiltered. The methylene chloride was evaporated resulting in an oilwhich was dissolved in 1N HCl (200 ml) and extracted with ether (100ml). The ether layer was discarded and the aqueous layer was made basicusing solid K₂ CO₃. The product was extracted into methylene chloridedried over Na₂ SO₄ and evaporated to give diethyl-3-aminopimaleate (7.5g). ¹ H NMR (d₆ -DMSO) δ1.25 (t, 3H, J =7 Hz), 1.26 (t, 3H, J=8 Hz),1.45 (m, 2H), 1.7 (m, 2H), 2.01 (bs, 2H), 2.45 (m, 2H), 3.2 (m, 1H),4.13 (q, 4H, J=8 Hz); MS (FAB) m/e 132.1 (M+H+) 186.2.

Step 2. Preparation of(±)-diethyl-3-[[4-[[4-(aminoiminomehtyl)phenyl]amino]-1,4-dioxobutyl]amino]heptanedioate

[[4-[[4-(Aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid hydrochlorideprepared in Example 1 (5.0 g, 18.5 mmol) was added to dry DMF (250 ml)followed by N-methylmorpholine (2.2 g, 18.5 mmol) and isobutylchloroformate (2.7 g, 18.5 mmol) at 25° C. The mixture was stirred for 5minutes. Diethyl-3-aminopimaleate (4.25 g, 18.5 mmol; from Step 1) wasadded followed by dimethylaminopyridine. After 1 hour, the solvent wasremoved under reduced pressure and the product purified by reverse phasechromatography (water/acetonitrile) to result in 4.1 g of a white solid:¹ H NMR (d₆ -DMSO) δ1.15 (t, 3H, J=7.3 Hz), 1.16 (t, 3H, J=8 Hz), 1.4(m, 2H), 2.50 (t, 2H, J=7.1 Hz), 2.49 (m, 4H), 2.58 (t, 2H, J=7.1 Hz),4.04 (m, 5H), 7.78 (s, 4H), 7.79 (d, 1H, J=12.4 Hz), 8.95 (bs, 2H), 9.15(bs, 2H), 10.40 (s, 1H), MS (FAB) m/e 449.0 (M+H+).

Elemental Analysis Required for C₂₂ H₃₂ N₄ O₆.F₃.C₂ O₂ H.H₂ O: C, 50.44;H, 5.95; N, 9.80. Found: C, 50.33; H, 6.02; N, 9.67.

In-Vitro Platelet Aggregation in PRP

The platelet-binding for activity of the compounds of the presentinvention can be demonstrated by the assay described below.

Healthy male or female dogs were fasted for 8 hours prior to drawingblood; then 30 ml whole blood was collected using a butterfly needle and30 cc plastic syringe with 3 ml of 0.129M buffered sodium citrate(3.8%). The syringe was rotated carefully as blood was drawn to mix thecitrate. Platelet-rich plasma (PRP) was prepared by centrifugation at975×g for 3.17 minutes at room temperature allowing the centrifuge tocoast to a stop without braking. The PRP was removed from the blood witha plastic pipette and placed in a plastic capped, 50 mL Corning conicalsterile centrifuge tube which was held at room temperature. Plateletpoor plasma (PPP) was prepared by centrifuging the remaining blood at2000×g for 15 minutes at room temperature allowing the centrifuge tocoast to a stop without braking. The PRP was adjusted with PPP to acount of 2-3×10⁸ platelets per mL. 400 uL of the PRP preparation and 50uL of the compound to be tested in solution or saline were preincubatedfor 1 minute at 37° C. in an aggregometer (BioData, Horsham, PA). 50 uLof adenosine 5' diphosphate (ADP) (50 um final concentration) was addedto the cuvettes and the aggregation was monitored for 1 minute. Allcompounds are tested in duplicate. Results are calculated as follows:Percent of control=[(maximal OD minus initial OD of compound) divided by(maximal OD minus initial OD of control saline)]×100. The %inhibition=100-(percent of control).

IC₅₀ 's (dosage at which 50% of platelet aggregation is inhibited) werecalculated by linear regression of the dose response curve. The assayresult for the compound of Example 3 was 4.8 μm.

What is claimed is:
 1. A compound selected from the group consisting of##STR22##